C(RGDyK)-mediated Pluronic-PBCA Nanoparticles Detoxification Of Central Organophosphate Poisoning

Objective: With the development of society,the shadow of war has always threatened the survival of human beings,especially weapons of mass destruction such as chemical weapons,biological weapons,nuclear weapons,etc.Nerve poisons and common organophosphorus pesticides are both organophosphorus poisons,which are very toxic and have a high usage rate.They can quickly pass through the skin,mucous membranes,gastrointestinal tract,etc.,which can be absorbed by the body and cause systemic poisoning,which can lead to multiple organ failure and death.Anticholinergic drugs are used for first aid and symptomatic treatment of neurotoxic poisoning,and the most fundamental means of pharmacological intervention is to separate the molecules of the agent from acetylcholinesterase(AChE)in the shortest time,and then quickly restore the function of AChE to hydrolyze ACh.Standard treatments for organophosphorus(OP)compound poisoning include the use of oxime drugs as AChE reactivation antidote.However,the presence of the blood-brain barrier in the body prevents these drugs from being efficiently delivered to the central nervous system.Amifostine(HI-6)is a widely used reactivator.Due to its hydrophilic nature,it is difficult to penetrate BBB.Therefore,we hope that through the drug delivery system,a large amount of HI-6,which has a better reactivation effect,will be transmitted through the BBB in a large amount,and will be rapidly released in the center to activate the inhibited AChE.Methods: In this study,interfacial polymerization was used to construct c(RGDyK)cyclic peptide-conjugated HI-6 nanoparticles.The use of biodegradable and less toxic polybutyl cyanoacrylate(PBCA)ensures the safety of treatment.The material Pluronic P85 is used as a carrier because it has the advantages of inhibiting P glycoprotein efflux and improving drug solubility.We take the dosage of the main drug(HI-6),the amount of the carrier material n-butyl cyanoacrylate(?-BCA),and the amount of Pluronic P85 as the three factors to examine the optimization index of the nanoparticles.By comprehensively examining the normalization value(OD)of the encapsulation efficiency,drug loading and particle size of HI-6 nanoparticles,the star point design-response surface method was used to optimize the prescription preparation process.The encapsulation efficiency and drug loading of HI-6-loaded nanoparticles were measured by dialysis,and the nanoparticles were characterized by a laser particle size analyzer.In this study,rat brain astrocytes(AS)and brain endothelial cells(BCECs)will also be cultured.A bilayer non-contact BBB model in vitro will be established and the model will be evaluated.Permeation efficiency of free HI-6 and various nanoparticles;CCK-8 method was used to investigate the toxicity of free HI-6 and its nanoparticles to brain capillary endothelial cells;qualitative observation of BCECs on various fluorescent labels by laser confocal microscopy Nanoparticle uptake;tracking the distribution of fluorescent dye-labeled nanoparticles in mice by in vivo imaging;using ELLMAN method to measure mouse whole blood and brain enzyme reactivation rates.Investigate the in vivo and in vitro targeting of different nanoparticles.Results: In this study,Pluronic P85 and c(RGDyK)cyclic peptides were successfully linked for the first time,and c(RGDyK)modified centrally-targeted Pluronic-PBCA nanoparticles were prepared by interfacial polymerization.After formulation optimization,the average entrapment efficiency of the prepared HI-6 nanoparticles was 63.69%,the average drug loading was 6.2%,the average particle size was 147.7 nm(PDI=0.32),the zeta potential was-7.12 mV,and the shape was circular,evenly distributed.We have successfully established a bilayer cell in vitro BBB model.The TBB value of the established BBB model reaches 183?,which meets the model requirements.Compared with non-modified nanoparticles,c(RGDyK)modified nanoparticles have better effect on penetrating brain endothelial cells.Free HI-6 and its nanoparticles have low toxicity to endothelial cells.Confocal microscope observation of c(RGDyK)-modified nanoparticles has high fluorescence intensity,indicating that endothelial cells take up modified nanoparticles more significantly than unmodified nanoparticles.In vitro in vivo imaging experiments show that nanoparticles modified with c(RGDyK)can more easily reach the brain to achieve central targeting in mice.By calculating the reactivation rates of the central and brain enzymes of the stained mice,it was found that the nanoparticle modified with c(RGDyK)has a stronger targeting effect on the brain than the non-modified one.Conclusion: In vitro and in vivo experiments have shown that c(RGDyK)modified nanoparticles have a stronger targeting effect on the brain than non-modified nanoparticles.In addition,the evaluation of reactivation shows that the modified nanoparticles have a higher reactivation rate for poisoned mice,which can reflect that the nanoparticles modified with cyclic peptide are more centrally targeted.The successful implementation of this study is expected to improve the treatment level of neurotoxic agents.